Study Of The Effects Of Zoledronic And Genistein On Bone Metabolism In OPG Gene-knockout Mice

Part one Changes of bone mineral density, microarchitecture andbiomechanical property in OPG - knockout miceObjective To study the changes of bone mineral density (BMD),microarchitecture and biomechanical property in osteoprotegerin(OPG)-knockout mice (OPG^-/-).Methods 10-week-old female OPG^-/- mice (n=10) and wild-type mice(n=10) were involved in the study. The trabecular and cortical bonemicroarchitecture was assessed by micro-CT in the right proximal tibia.BMD of the left femur was detected by DXA. The biomechanicalproperty of the right femur was determined by a three-point bending test.Serum biochemical markers of bone turnover such as bone alkalinephosphatase(B-ALP),tartrate-resistant acid phosphatase-5b (TRACP-5b),and receptor activator of nuclear factorκB ligand (RANKL) weredetermined by ELISA.Results Compared with wild-type mice, the OPG^-/- mice showed adecreased volumetric BMD (vBMD),tissue BMD(tBMD), bone volumefraction (BVF), bone surface fraction(BSF), trabecular number (Tb.N),and trabecular connectivity density (Conn.D), with an increased structuremodel index(SMI), trabecular thickness(Tb.Th) and trabecular separation(Tb.Sp). The cortical bone microarchitecture parameters such as corticalthickness (Ct.Th), cortical area (Ct.Ar), total area (Tt.Ar), Moment ofInertia (Mm), cortical BMD (Ct.BMD) and cortical bone mineral containt(Ct.BMC) were also decreased, with an increased inner perimeter(In.Pm) and marrow area (Ma.Ar). Three-point bending test revealed adecrease in ultimate load, ultimate stress, stiff index and elastic modulus.The BMD was reduced by DXA determination, while serum B-ALP,TRACP-5b, and RANKL were all elevated at the same time.Conclusions The turnover rate of OPG knockout mice is high withdramatic disturbances in bone microarchitecture and deterioration in bonebiomechanical property. Part two In vivo studies of zoledronic acid on the mechanism ofanti-bone resorptionobjective To study the in vivo anti-bone resorption mechanism ofzoledronic acid.Methods Six-week-old female OPG^-/- mice (n=20) and wild-type mice(n=10) were involved in the study. OPG^-/- mice were randomly dividedinto two groups(10 for each group), and treated with zoledronic acid at adose of 0μg/kg (H₂O group) and 150μg/kg (Zol group) subcutaneouslytwice per week. The mice were killed 6 weeks after intervention.Trabecular and cortical bone microarchitecture in the right proximal tibiawas assessed by micro-CT and the bone mineral density (BMD) of theleft femur was measured by DXA. The biomechanical property of theright femur was determined by a three-point bending test. Serum bonealkaline phosphatase (B-ALP), tartrate-resistant acid phosphatase-Sb(TRACP-Sb) and receptor activator of nuclear factorκB ligand (RANKL)were determined by ELISA.Results Compared with the mice in H₂O group, the zoledronicacid-treated mice showed an increased total BMD and BMC, almostequal to the levels of wild-type mice.μCT analysis revealed an increasein vBMD, tBMD, BVF, BSF, Tb.Th,Tb.N and Conn.D, with a decreasein SMI and Tb.Sp; the cortical bone microarchitechitecture parameterssuch as Ct.Th, Mm, Ct.Ar, Tt.Ar, Ct.BMD and Ct.BMC were allincreased, with an decreased In.Pm and Ma.Ar. Three-point bending testshowed an increase in ultimate load, ultimate stress, elastic modulus andstress index. A decrease in serum TRACP-Sb and an increase in serumRANKL were revealed by ELISA analysis.Conclusions Zoledronic acid could effectively prevent bone lose in mice lacking osteoprotegerin, suggesting that the in vivo anti-boneresorption effect of zoledronic acid is triggered via an OPG-independentpathway, possibly by direct suppression of the activity of osteoclasts.Part three Studies of genistein effects on bone metabolism in OPG^-/-miceObjective To study genistein in vivo effects on bone metabolism inOPG^-/- miceMethods Following heterozygotes(OPG^+/-) matings, thehomozygotes(OPG^-/-)and wild type(WT)with a mixed C57BL/6J×129/SVbackground were obtained. 6-week-old female OPG^-/- mice (n=40) andwild-type mice (n=8) were involved in the study. OPG^-/- mice wererandomly divided into 5 groups (n=8 for each group): 1) Genistein-treatedmice: treated with genistein at a maximal dose(0.8mg/day)subcutaneously(Gen); 2) 17β-Estradiol-treated mice: treated with17β-Estradiol at a maximal dose(0.034μg/day) subcutaneously(E₂); 3)DMSO-treated mice: treated with a mixture of dimethylsulfoxide(DMSO) and polyethylenglycol-300 subcutaneously; 4) Zoledronicacid-treated mice: treated with zoledronic acid at a dose (150μg/kg)twice per week subcutaneously (Zol); 5) H₂O-treated mice: treated withsterilized water twice per week subcutaneously. The mice were killed 6weeks after intervention. Trabecular and cortical bone microarchitecturewas assessed by micro-CT in the right proximal tibia. The bone mineraldensity (BMD) of the left femur was measured using DXA. Thebiomechanical property of the right femur was determined by athree-point bending test. Serum bone alkaline phosphatase (B-ALP),tartrate-resistant acid phosphatase-5b (TRACP-Sb) and receptor activator of nuclear factorκB ligand (RANKL) were determined by ELISA.Results DXA analysis revealed that the total BMD of femur was notsignificantly changed in Gen, E₂, H₂O, and DMSO groups. Three-pointbending test showed no significant differences in biomechanicalparameters including ultimate load, ultimate stress,stiff index, and elasticmodulus, and micro-CT analysis showed that trabecular bonemicroarchitectural parameters (vBMD,tBMD,BVF,BSF,SMI,Tb.N,Conn.D,Tb.Sp and Tb.Th) and cortical bone microarchitecturalparameters(Ct.Th,Mm,In.Pm,Ot.Pm,Ma.Ar,Ct.Ar,Tt.Ar,Ct.BMDand Ct.BMC) were not different either in these groups. Genistein and17β-Estradiol did not modify either serum TRACP-5b or B-ALP orRANKL levels. However, in addition to increases of bone mass,zoledronic acid could effectively improve biomechanical property andprevented the architectural bone from deteriorating in OPG^-/- mice.Conclusions Lack of osteoprotegerin in mice, the effects of genisteinas well as 17β-Estradiol on bone metabolism in vivo disappeared,suggesting that their activity on bone metabolism should be totallyOPG--dependent.Part four Estabolishment of osteoprotegerin transgenic mice.Objective osteoprotegerin(OPG) may play a key role in the regulationof bone metabolism. In order to evaluate its effects in vivo, wedeveloped an OPG transgenic mouse model.Method Expression plasmid pCI-hOPGp-mOPG containing humanOPG promoter was successfully reconstructed, and micro-injected intofertilized zygotes from C57BL/6J×CBA mice to prepare trangenic mice.Progeny were screened for the presence of the transgene by PCR. Resuts Out of the 69 founder generation, 7 founder mice wereidentified by PCR dectetion of OPG. Out of the 120 F₁ generation, 15OPG transgenic mice were obtained.Conclusion Four OPG transgenic mice lines have been developed,which could be valuable for studying the biological significance and generegulation of OPG in vivo.